The orally active antihyperglycemic drug beta-guanidinopropionic acid is transported by the human proton-coupled amino acid transporter hPAT1.

The orally administered creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a beta-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of beta-GPA is well established, the underlying uptake mechanism has not yet been studied. We investigated whether the H(+)-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Uptake of l-[(3)H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by beta-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants of about 1.5 mM. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that beta-GPA, GAA and 4-GBA are effectively transported by this transport system in an electrogenic manner. We conclude that hPAT1 might be responsible for the intestinal absorption of beta-GPA thereby allowing its oral administration. Moreover, with beta-GPA we identified a new high affinity hPAT1 substrate that might be an interesting starting point for future drug design-drug delivery strategies.

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Transport of valproate at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells: mechanism and substrate specificity.

To reach its target cells, the antiepileptic drug valproate has to cross both the intestinal epithelial barrier and the blood-brain barrier in intact form as well as in sufficient amounts. This study was performed to characterize the epithelial transport of valproate at intestinal (Caco-2) and at blood-brain barrier (RBE4) cells. At both cell types, uptake of [(3)H]valproate was independent of inwardly directed Na(+), Ca(2+), Mg(2+), K(+) or Cl(-) gradients. Uptake was, however, strongly stimulated by an inwardly directed H(+) gradient. The cells accumulated valproate against a concentration gradient and the uptake rate of valproate was saturable with K(t) values of 0.6 and 0.8mM. At Caco-2 cell monolayers, the total apical-to-basolateral flux of [(3)H]valproate exceeded the basolateral-to-apical flux 14-fold. Various monocarboxylic acids like salicylate, benzoate, acetate, propionate, butyrate, hexanoate, diclofenac and ibuprofen inhibited [(3)H]valproate uptake at both cell types. Lactate and pyruvate inhibited valproate uptake at RBE4 cells but not at Caco-2 cells. We conclude that valproate is accumulated in intestinal cells against a concentration gradient by the activity of a specific H(+)-dependent DIDS-insensitive transport system for monocarboxylates not identical with monocarboxylate transporter 1 (MCT1). The passage of valproate across the blood-brain barrier is very likely mediated by MCT1.

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1.

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.

Influence of a proton gradient on the transport kinetics of the H+/amino acid cotransporter PAT1 in Caco-2 cells.

The recently cloned proton-coupled amino acid transporter 1 (PAT1) not only accepts several amino acids as substrates but also pharmaceutically relevant L-proline or GABA derivatives such as cis-4-hydroxy-L-proline, L-azetidine-2-carboxylic acid (LACA), 3-amino-1-propanesulfonic acid, nipecotic acid, and the antituberculotic agent D-cycloserine. Because human intestine expresses hPAT1 at the brush border membrane, the transporter may serve as a new oral drug delivery route. Using the human intestinal cell line Caco-2, we have investigated the influence of an inwardly directed proton gradient on the kinetic parameters of L-proline uptake. H+ altered only the apparent affinity of L-proline transport and not the maximal transport velocity. Similarly, treatment of the cells with diethylpyrocarbonate (DEPC), known to chemically modify histidyl residues and block their function, affected only the Kt value of L-proline transport. Both increasing pH and DEPC treatment strongly increased the inhibition constants (Ki) of several drugs at hPAT1. It is concluded that H+ stimulates hPAT1 primarily by increasing the substrate affinity with no detectable influence on the maximal transport velocity of the transporter.

Substrate specificity and mechanism of the intestinal clonidine uptake by Caco-2 cells.

PURPOSE: This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. METHODS: Uptake of [3H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. RESULTS: Uptake of [3H]clonidine was linear for up to 2 min, Na+-independent, and insensitive to changes in membrane potential, but strongly H+-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5+/-0.1 mM (Kt) and 16.6+/-1.8 nmol/2 min per mg of protein (Vmax) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [3H]clonidine uptake with Ki values between 0.15 and 1 mM. CONCLUSIONS: Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood-brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H+/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.

Drug specificity and intestinal membrane localization of human organic cation transporters (OCT).

This study was performed to investigate which human organic cation transporter, hOCT1, hOCT2 or hOCT3, participates with regard to cation specificity and membrane localization in the intestinal absorption of orally available cationic drugs. Inhibition of N-[methyl-3H]4-phenylpyridinium ([3H]MPP+) uptake by various compounds into Caco-2 cells and into cells (HEK-293 or CHO) that were stably transfected with hOCT1, hOCT2 or hOCT3 was compared. The uptake of [3H]MPP+ into Caco-2 cells was inhibited by atropine, butylscopolamine, clonidine, diphenhydramine, etilefrine, quinine and ranitidine with IC50 values between 6 microM and 4 mM. Transepithelial, apical to basal flux of [3H]MPP+ across Caco-2 cell monolayers was also strongly inhibited by these compounds. The inhibitory potency of the cationic drugs and prototypical organic cations at Caco-2 cells correlated well with the inhibitory potency measured at CHO-hOCT3 cells but much less with that at HEK-hOCT1 and -hOCT2 cells. This is functional evidence for the predominant role of hOCT3. Etilefrine and atropine were specifically transported into CHO cells by hOCT3. In Caco-2 cells, the mRNA of all three hOCT and the proteins hOCT2 and hOCT3 were detected. More importantly, immunocytochemical analyses of human jejunum revealed for the first time that hOCT3 is localized to the brush border membrane whereas hOCT1 immunolabeling was mainly observed at the lateral membranes of the enterocytes.

Serotonin, L-tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1.

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.1 mM. Uptake of l-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by l-proline. Whereas PAT1 substrates produced significant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, l-tryptophan, tryptamine, and 5-hydroxy-l-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with l-tryptophan, tryptamine, 5-hydroxy-l-tryptophan, or serotonin. Although not transported electrogenically by PAT1, l-tryptophan and its derivatives inhibited glycine-evoked currents dose-dependently. We conclude that serotonin, l-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers' naturally occurring inhibitors that may alter the transport function of PAT1.

Transport of pharmacologically active proline derivatives by the human proton-coupled amino acid transporter hPAT1.

Several proline derivatives such as L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, and 3,4-dehydro-DL-proline prevent procollagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors. This study was performed to investigate whether the recently discovered human proton-coupled amino acid transporter 1 (hPAT1) is capable of transporting such pharmacologically relevant proline derivatives and also GABA analogs. Uptake of L-[3H]proline and [3H]glycine in Caco-2 cells was Na+-independent but strongly H+-dependent. The L-proline uptake was saturable and mediated by a single transport system (hPAT1) with an affinity constant of 2.0 +/- 0.2 mM. The uptake of L-[3H]proline was inhibited by D-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, 3,4-dehydro-DL-proline, L-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, D- and L-pipecolic acid, l-thiaproline, and many others. Apical uptake and transepithelial flux of L-[3H]proline across Caco-2 cell monolayers were strongly inhibited by proline derivatives in proportions corresponding to their respective affinity constants at hPAT1. The basolateral to apical flux of L-[3H]proline was only 8% of that in the opposite direction. Apical uptake of unlabeled L-proline, cis-4-hydroxy-L-proline, and L-azetidine-2-carboxylic acid was stimulated by an inside directed H+ gradient 2- to 3-fold. Total apical to basolateral flux of proline derivatives was moderately correlated with their inhibitory potency for L-[3H]proline uptake and flux inhibition. We conclude that 1) the substrate specificity of hPAT1 is very much broader than so far reported and 2) the system accepts therapeutically relevant proline and GABA derivatives. hPAT1 is a promising candidate for new ways of oral drug delivery.